Contrast Agents for Magnetic Resonance Imaging

ABSTRACT

Ascorbate or a pharmaceutically acceptable salt thereof is described for use in carrying out a method, or for the preparation of a medicament for carrying out a method, of enhancing a magnetic resonance imaging (MRI) image of a body or body region such as an organ or organ region in a subject. The method is carried out by parenterally administering ascorbate or a pharmaceutically acceptable salt thereof to the subject in an MRI image-enhancing amount; and then generating, by MRI of the subject, an image of the body or body region. The ascorbate or pharmaceutically acceptable salt thereof enhances the MRI image.

RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. 111(a) ofPCT/US2016/054478, filed Sep. 29, 2016, which in turn claims the benefitof U.S. Provisional Patent Application Ser. No. 62/234,986, filed Sep.30, 2015, and U.S. Provisional Patent Application Ser. No. 62/291,138,filed Feb. 4, 2016, the disclosure of each which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present invention concerns compositions useful as contrast agentsfor magnetic resonance imaging and methods of use thereof.

BACKGROUND OF THE INVENTION

Magnetic resonance imaging (MRI) produces exquisite renderings of humananatomy and pathology at high spatial resolution. To increase diagnosticsensitivity and specificity for MRI, such as with imaging for cancer,infection, neurological and heart diseases, contrast material is oftenadministered intravenously before and/or during imaging to improvesignal.

The most common MRI contrast material is based on molecular complexescontaining the paramagnetic metal gadolinium (Gd). In the U.S., all nineFDA-approved MRI contrast agents are Gd-based. Gd possesses strong“paramagnetism” that results in a locally increased MRI signal onT₁-weighted images. However, Gd-based contrast agents can cause a rarebut severely debilitating condition called nephrogenic systemic fibrosis(NSF), a syndrome involving widespread fibrosis of the skin, joints,eyes, and internal organs. The WHO and FDA have issued restrictions onthe use of these agents in patients with renal insufficiency/failure,with the FDA mandating a “black box” warning on all commercial mediacontaining gadolinium. As a consequence, millions of patients in theU.S., and many more worldwide, are no longer able to receive contrastmaterial for MRI, severely limiting detection and characterization forseveral diseases.

Other paramagnetic complexes, used more rarely either as investigationalor as “off-label,” are usually based on large iron oxide-basednanoparticles developed and marketed as intravenous iron replacementtherapy (e.g., FERAHEME® (ferumoxytol) injection). The use of thesecomplexes for MRI is limited, however, by their large molecular size,which confines these agents to the blood pool until they are finallycleared by the reticuloendothelial system (i.e., macrophages, liver,spleen).

U.S. Patent Application Publication 2014/0154185 to Van Zijl et al.discusses the use of parenteral glucose to enhance MRI. See also Yadav NN, Xu J, Bar-Shir A, Qin Q, Chan K W, Grgac K, Li W, McMahon M T, vanZijl P C, Natural D-glucose as a biodegradable MRI contrast agent fordetecting cancer. Magn Reson Med. 2012 December; 68(6):1764-73; Yadav NN, Xu J, Bar-Shir A, Qin Q, Chan K W, Grgac K, Li W, McMahon M T, vanZijl P C, Natural D-glucose as a biodegradable MRI relaxation agent.Magn Reson Med. 2014 September; 72(3):823-28.

There remains a need for alternative contrast agents useful for MRIscanning technologies.

SUMMARY OF THE INVENTION

Provided herein are methods useful for magnetic resonance imaging (MRI)using parenteral ascorbate (Vitamin C) as a contrast agent for thedetection and characterization of perfusion, metabolism, and oxidativestress in human and non-human tissues without the need for radioactivityor chemical labeling. After parenteral administration, time-dependentmagnetic resonance (MR) signal changes are detected in tissues whereascorbate is taken up and/or passes through. These MRI signal changesare detectable using routine spin echo or gradient echo-basedT₂-weighted MRI sequences and quantifiable with T₂ mapping. Other lesscommon acquisition techniques sensitive to spin-spin relaxation may alsobe used to encode MR signals.

Thus, provided herein is a method of enhancing an MRI image of a body orbody region, such as an organ or organ region in a subject, which methodincludes parenterally administering (e.g., intravenous, intraperitoneal,intraarterial, intraosseous, or intrathecal administration) ascorbate ora pharmaceutically acceptable salt thereof to said subject in an MRIimage-enhancing amount; and then generating, by MRI of the subject, animage of said body or body region, whereby the ascorbate orpharmaceutically acceptable salt thereof enhances the MRI image.

In some embodiments, the MRI image is generated during, or up to 5, 10,30, 40, 60, 90 or 120 minutes after, the parenterally administering ofascorbate or pharmaceutically acceptable salt thereof.

In some embodiments, the ascorbate or pharmaceutically acceptable saltthereof is sodium ascorbate, meglumine ascorbate, or a mixture thereof(e.g., meglumine ascorbate and sodium ascorbate in a molar or millimolar(mM) ratio of from 10:90, 20:80, 30:70, or 40:60, up to 90:10, 80:20,70:30, or 60:40 (meglumine ascorbate:sodium ascorbate)).

Further provided is the use of ascorbate or a pharmaceuticallyacceptable salt thereof for carrying out a method as taught herein, orfor the preparation of a medicament or imaging agent for carrying out amethod as taught herein.

The present invention is explained in greater detail in the drawingsherein and the specification set forth below. The disclosures of allUnited States patent references cited herein are to be incorporated byreference herein in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. Ascorbate and dismutation of the ascorbate radical. FIG.1A, Ascorbate is a di-acid, however at pH 7.4, 99% is present as themono anion (AscH⁻). Ascorbate radical (Asc.⁻) is present at equilibrium(but also at much lower concentrations) with oxidized and reduced formsof ascorbate. FIG. 1B, The dismutation of Asc.⁻ is the principal routeof its transformation, with a rate constant (k_(obs)) that falls intothe “intermediate” proton exchange rate on the NMR timescale. This rateconstant can increase by a factor of 10 in the presence of phosphate(Bors W, Buettner G R. (1997) The vitamin C radical and its reactions inVitamin C in Health and Disease, ed. by L. Packer and J. Fuchs, MarcelDekker, Inc., New York, Chapter 4, pp75-94).

FIG. 2. T₂ relaxivity (r₂=mM⁻¹ sec⁻¹) of sugars, sugar alcohols, andascorbate Comparisons include both mono and disaccharides. As discussedin the text, note the diminishing contrast effect at higherconcentration, believed secondary to self-association of like moietiesand reduced proton exchange.

FIGS. 3A-3D. In vitro (“phantom”) experiments on ascorbate spin-spinrelaxation (T₂-weighted) MM contrast. FIG. 3A, shows quantitative T₂mapping in 5 phantoms with progressively increasing ascorbate (AA)concentration. Statistically significant “negative T₂ contrast” (signalloss) is seen as low 1-5 mM as compared to control (phosphate-bufferedsaline) with conventional fast spin echo (FSE) acquisition. Sensitivityis therefore at the lower end of expected tissue/cellular concentrationsfollowing pharmacological doses of ascorbate in high uptake tissues(e.g., tumors and brain, 10-30 mM). This also does not include anysynergistic effect from tissue oxidative substrates or exchangecatalysts. FIG. 3B, shows the synergistic effect of H₂O₂ on ascorbate T₂enhancement. H₂O₂, which rises to 100-200 micromolar in brain and tumorsin vivo following parenteral ascorbate, produces a marked synergism onthe T₂ contrast effect from ascorbic acid. The synergistic effect slowlydiminishes over time in phantoms over 30 min as above, but will besustained in vivo as long as H₂O₂ is produced following pharmacologicalascorbate doses. FIG. 3C, demonstrates the influence of pH onascorbate's T₂ effect, which is maximized at neutral/physiological pH(7.0-7.4). This result is consistent with prior studies on the ratekinetics of ascorbate disproportionation with its radical and oxidizedform at equilibrium (FIGS. 1A-1B). FIG. 3D, reveals a marked synergisticeffect when ascorbate (Asc) is salified (salted) with meglumine (Meg),an amine sugar derivative of sorbital that is commonly employed as anexcipient in several FDA-approved drug and contrast formulations.

FIG. 4. Comparison of Ascorbate as Na or Meglumine Salt (Meg). Solutionsare prepared with physiological concentrations of PO₄ (2 mM) and HCO₃ ⁻(25 mM) buffers.

FIG. 5. Na Ascorbate (Asc)+Physiological Exchange Catalysts. Eachsolution is set at neutral (pH=7.0) in deionized water. Concentrationsof physiological exchange catalysts are the same as the in vivo in serumand extracellular space: PO₄=2 mM, glucose=5 mM, and HCO₃ ⁻ is 25 mM.

FIG. 6. Exchange Synergism Between Na Ascorbate (Na Asc)/Meglumine (Meg)and Glucose (Glu). Solutions are compared in the setting ofphysiological buffers PO₄ (2 mM) and HCO₃ ⁻ (25 mM) that also contributeas exchange catalysts.

FIG. 7. Exchange synergism of Na Asc/Meglumine (Meg) with sugaralcohols, mono- and disaccharides. All solutions are prepared inco-presence of 2 mM PO₄ and 25 mM HCO₃ ⁻.

FIG. 8. Resealed data without control for exchange synergism of NaAsc/Meglumine (Meg) with sugar alcohols, mono- and disaccharides. Allsolutions are prepared with 2 mM PO₄ and 25 mM HCO₃ ⁻.

FIGS. 9A-9B. In vivo ascorbate T2 contrast changes following high doseparenteral ascorbate (2 g/kg, right IJ iv injection.) FIG. 9A, shows aconventional single slice axial FSE T2WI image through the midbrain of anormal C57 black mouse, and the two images on the right demonstrate a‘first pass’ extraction of contrast change during and followingascorbate administration i.v. T2 signal in brain tissue immediatelyfollowing, and 10 minutes after, ascorbate administration is acquiredand then subtracted from the T2 brain signal acquisition pre ascorbateadministration. Since ascorbate produces a decrease in signal intensity,subtraction from the higher signal pre-dose scan results in a netpositive ‘map’ of flow-through perfusion (blood flow) through braintissue. At 10 minutes, the perfusion effect has nearly resolved andearly signal intensity changes related to tissue uptake are beginning tobe observed. FIG. 9B, show the signal changes due to tissue uptake ofhigh-dose ascorbate. Color-lute maps of signal intensity are notsubtracted from the pre-dose scan and therefore show the expecteddecreases in T2 signal over time, maximized between 30-60 min in normalC57 mice.

FIG. 10. Ascorbate T2 enhancement in a rodent model of neocorticalspreading depression. In the illustrated experiment, the lower row ofimages shows a tiny craniotectomy with gelfoam (red arrow) soaked in ahigh concentration of KCl, which diffuses locally into the adjacentparietal cortex. The craniectomy site is 1 mm posterior to bregma, askull landmark representing the posterior third of the underlying brain.The above two rows show T2 images and quantitative color lute T2 maps ofsignal change in rodent brain that are 3 and 4 mm anterior to bregma,that is, distant from the SD induction site. T2 signal changes in theanterior slices demonstrate clear T2 asymmetry in the right cerebralcortex as compared to the left (again, SD remains confined to the righthemisphere). These marked cortical signal changes are consistent withthe known hypermetabolic activity that occurs with SD, as also observedwith ¹⁸F-FDG PET, and with direct microdialysis and metabolomicdeterminations. Of note, the opposite observation (focally increased T2signal) is seen directly under the ectom site itself (row three),consistent with localized edema (increased free water) at the site ofKCl infusion.

FIGS. 11A-11B. Perfusion and viability cardiac imaging with parenteralascorbate. FIG. 11A depicts the two primary imaging planes, coronal andaxial, for rat heart imaging at 7T. FIG. 11B shows transient decrease inT2 signal intensity throughout the left ventricle with initial bolus ofascorbate injection i.v.

FIGS. 12A-12C. T2 contrast changes in guinea pigs following i.v.administration of three different formulations of ascorbate. FIG. 12A,Fast spin echo (FSE) T2 images before and after 60 min slow infusion ofascorbate show dramatic signal intensity differences throughout thebrain parenchyma. FIG. 12B and FIG. 12C respectively show normalizedsignal intensity changes and quantitative relaxivity measurements areshown for both guinea cerebral cortex (Cx) and basal ganglia (BG) afteradministration of three different ascorbate formulations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Subjects for the present invention are, in general, mammalian subjects,including both human subjects and animal subjects (e.g., dogs, cats,rabbits, cattle, horses, etc.) for research or veterinary purposes.Subjects may be male or female and may be any age, including neonate,infant, juvenile, adolescent, adult, and geriatric subjects.

Magnetic resonance imaging (MRI) is known, and may be carried out bycommercially available equipment, and by techniques known in the field.See, e.g., S. Bushong and G. Clarke, Magnetic Resonance Imaging:Physical and Biological Principles (Mosby, 4^(th) Ed. 2014). In someembodiments, the MRI is perfusion (e.g., blood flow) imaging. In someembodiments, the MRI is metabolism imaging. Metabolism imaging may beused as a diagnostic biomarker analogous to 18F FDG PET, including, butnot limited to, identification/characterization of tumors ordysfunctional tissues demonstrating hyper- or hypo-metabolism.

“Body or body region” that may be imaged with MRI as taught hereinincludes the body or any region of the body of a subject, such as anorgan or organ system, soft tissue, bone, etc., or any portion thereof.Examples of body regions include, but are not limited to, head, neck,thorax, abdomen, pelvis, limb(s), muscle, fat, other soft tissues, bone,etc. Examples of organs include, but are not limited to, adrenal gland,pituitary, thymus, corpus luteum, retina, brain, spleen, lung, testicle,lymph nodes, liver, thyroid, small intestinal mucosa, leukocytes,pancreas, kidney, salivary gland tissue, heart, etc.

“Enhancing” an MRI image as used herein is inclusive of facilitating theMRI visualization by enhancing the contrast of structures or fluids inan MRI signal.

An “MRI contrast agent” is a substance that can enhance the contrast ofstructures or fluids within the body during an MRI scan. Examplesinclude, but are not limited to, paramagnetic contrast agents such asgadolinium(III) containing agents or manganese chelates, andsuperparamagnetic agents such as iron platinum particles. See also U.S.Patent Application Publication Nos. 2014/0350193 to Axelsson et al.;2014/0234210 to Lin et al.

“Parenteral administration” as used herein includes, but is not limitedto, intravenous, subcutaneous, intramuscular, intraperitoneal,intraarterial, intraosseous, or intrathecal administration, e.g.,through injection or infusion. As a non-limiting example,intraperitoneal or other parenteral administration may be used whereintravenous (i.v.) access is difficult for a subject (e.g., low bloodpressure), or the route of administration otherwise would result in asuitable MRI image.

MR Imaging and Clinical Application of Contrast Media.

Clinical magnetic resonance imaging (MRI) generates high-resolutionimages of the body through the acquisition of proton (¹H) NMR signalsfrom water and macromolecules in tissue. For “T₁-weighted” MR images,signal intensity increases in regions where longitudinal relaxation rate(spin lattice relaxation rate, 1/T₁) increases. With “T2-weighted” MRI,signal intensity decreases when transverse relaxation rate (spin-spinrelaxation rate, 1/T₂) increases. Both T₁ and T₂ weighted images areroutinely acquired in virtually all clinical MRI studies.

Intravenous contrast agents are routinely administered in MRI to furtherincrease 1/T₁ or 1/T₂, in an effort to better delineate diseased fromnormal tissue, improve anatomical definition, and enhancecharacterization of physiological or pathological function. Almost allcurrently approved MRI contrast agents are based on chelates of thelanthanide metal Gadolinium (Gd), with a smaller subset of angiographicand perfusion studies conducted using iron-oxide materials (e.g.,Feraheme) off-label in patients with renal insufficiency/failure.Commercial Gd-based materials are used most commonly to increase 1/T₁ indiseased tissue, where contrast material is prone to accumulate.

For tissue perfusion determinations with MRI, Gd-based agents oriron-oxide nanoparticles may be used, with acquisition strategies basedon either 1/T₁ or 1/T₂ contrast, although 1/T₂ contrast approaches areincreasingly favored. Perfusion imaging is currently used clinically tocharacterize tumor aggressiveness, tumor response to therapy, and tissueviability in heart, brain and other organs.

Ascorbic Acid.

Ascorbic acid (“L-ascorbic acid,” “ascorbate,” “Vitamin C”) is anaturally-occurring organic compound and an essential nutrient, withimportant properties as an antioxidant and co-factor in at least eightenzymatic reactions, including several collagen synthesis reactionsthat, when dysfunctional, result in the most conspicuous symptoms ofscurvy. Most mammals make ascorbic acid in the liver, where the enzymeL-gulonolactone oxidase converts glucose to ascorbic acid. In humans,higher primates, guinea pigs and most bats, however, a mutation resultsin low or absent L-gulonolactone oxidase expression, so that ascorbatemust be consumed in the diet (Lachapelle, M. Y.; Drouin, G. (2010).“Inactivation dates of the human and guinea pig vitamin C genes”.Genetica 139 (2): 199-207). In all animal species, L-ascorbicacid/ascorbate is the most abundant intracellular antioxidant, withintracellular concentrations capable of reaching 10-30 mM in tumors,brain cells, and some other tissues. Those tissues that accumulate over100 times the level in blood plasma of vitamin C include the adrenalglands, pituitary, thymus, corpus luteum, and retina. Those with 10 to50 times the concentration include brain, spleen, lung, testicle, lymphnodes, liver, thyroid, small intestinal mucosa, leukocytes, pancreas,kidney, and salivary glands (Hediger M A (May 2002). “New view at C”.Nat. Med. 8 (5): 445-6).

Dietary excesses of vitamin C are not absorbed, and excesses in theblood are rapidly excreted in the urine. Vitamin C exhibits remarkablylow toxicity, with an LD₅₀ in rats generally accepted at ˜11.9 grams perkilogram of body weight by forced gavage. The mechanism of death fromsuch doses (1.2% of body weight, or 0.84 kg for a 70 kg human) isunknown, but may be mechanical rather than chemical (“Safety (MSDS) datafor ascorbic acid”. Oxford University. Oct. 9, 2005. Retrieved Feb. 21,2007). The LD₅₀ in humans is uncertain given the lack of any accidentalor intentional poisoning death data. The rat LD₅₀ is therefore used as aguide for human toxicity.

At physiological pH, 99% of ascorbic acid is present as the mono anion(FIG. 1A). The chemistry, and therefore imaging properties, of vitamin Care dominated by this moiety. As a donor antioxidant, the mono aniondonates a hydrogen atom (H. or H⁺+e⁻) to an oxidizing radical to producea resonance-stabilized tricarbonyl ascorbate free radical, Asc.⁻ (FIG.1B). The dismutation reaction of Asc.⁻ back to reduced or oxidizedascorbate is the principal route of elimination in vitro. This processis supplemented in vivo by enzymes that aid in ascorbate recycling (MayJ M, Qu Z C, Neel D R, Li X (May 2003). “Recycling of vitamin C from itsoxidized forms by human endothelial cells”. Biochim. Biophys. Acta 1640(2-3): 153-61). Dismutation of the radical to either ascorbate ordehydroascorbate occurs via loss or gain of hydrogen, which serves aseither the electron carrier or the more conventional cation. Also, therate constant of ascorbic radical dismutation is 10⁵-10⁶ M⁻¹ s⁻¹, sothat hydrogen exchange accompanying dismutation also occurs at the samerate. On the NMR timescale, these “intermediate” exchange rates areoptimal for altering ¹H spin-spin relaxation.

Ascorbate Transport and Excretion.

Ascorbic acid is absorbed in the body by both active transport andsimple diffusion. The two major active transport pathways aresodium-ascorbate co-transporters (SVCTs) and hexose transporters(GLUTs). SVCT1 and SVCT2 import the reduced form of ascorbate across theplasma membrane (Savini I, Rossi A, Pierro C, Avigliano L, Catani M V(April 2008). “SVCT1 and SVCT2: key proteins for vitamin C uptake”.Amino Acids 34 (3): 347-55), whereas GLUT1 and GLUT3 glucosetransporters transfer the oxidized form, dehydroascorbic acid (Rumsey SC, Kwon O, Xu G W, Burant C F, Simpson I, Levine M (July 1997). “Glucosetransporter isoforms GLUT1 and GLUT3 transport dehydroascorbic acid”. J.Biol. Chem. 272 (30): 18982-9). Although dehydroascorbic acidconcentrations are low in plasma under normal conditions, the oxidizedmolecule is absorbed at much higher rates across GLUT1 and 3 than thereduced form is across the SVCTs. When ascorbate concentrations arepharmacologically elevated, dehydroascorbate concentration alsoincreases, enabling marked absorption where GLUT transporters exist inhigh copy such as brain (and blood brain barrier) and tumor cells. Oncetransported, dehydroascorbic acid is rapidly reduced back to ascorbate.

Ascorbate concentrations over the renal re-absorption threshold passfreely into the urine and are excreted with a half-life of about 30minutes. At high dietary doses (corresponding to several hundred mg/dayin humans) the renal resorption threshold is 1.5 mg/dL in men and 1.3mg/dL in women (Oreopoulos D G, Lindeman R D, VanderJagt D J,Tzamaloukas A H, Bhagavan H N, Garry P J (October 1993). “Renalexcretion of ascorbic acid: effect of age and sex”. J Am Coll Nutr 12(5): 537-42). Ascorbate that is not directly excreted in the urine ordestroyed by other body metabolism is oxidized by L-ascorbate oxidaseand removed.

Without wishing to be bound by theory, the mechanism of ascorbate signalchange without paramagnetism, which is also described as “T₂-weightedcontrast,” is based on enhancement of the water proton (¹H) spin-spinrelaxation rate 1/T₂ (or reciprocally, spin-spin relaxation time, T₂),as solvent water protons are exchanged with hydroxyl protons onascorbate molecules. The effect of proton exchange on T₂ contrast isamplified further by the dismutation reaction of the ascorbate radicalat physiological pH. Ascorbate oxidation and ascorbate radicaldismutation are, in turn, driven by the co-presence of oxidizingsubstrates such as hydrogen peroxide (H₂O₂) and/or hydrogen (“proton”)exchange catalysts.

Ascorbate, especially in the presence of, and co-formulated with,spin-spin exchange catalysts (for example, simple sugars, sugar alcoholsor amino acids) is a safe and biodegradable MRI contrast agent thatrequires neither the use of metal-based (e.g., gadolinium or iron)contrast material nor ionizing radiation. The technique enablesassessment of tissue perfusion as well as high-resolution molecularcharacterization of tissue viability and metabolism that is analogous to¹⁸F-MG PET. The latter is possible by virtue of ascorbate's uptake (viadehydroascorbate) into cells through the same glucose transportmechanisms that take up ¹⁸F-FDG (i.e., GLUT 1 and 3 transporters)(Rumsey S C, Kwon O, Xu G W, Burant C F, Simpson I, Levine M (July1997). “Glucose transporter isoforms GLUT1 and GLUT3 transportdehydroascorbic acid”. J. Biol. Chem. 272 (30): 18982-9).

Potential applications for ascorbate MRI include several clinicalscenarios where PET scanning has already proven valuable but also whereimprovements in methodology will yield further clinical benefit. Thesescenarios include diagnostic studies for cancer, neurological disease(e.g., dementia, TBI and epilepsy) and cardiovascular imaging. Heartstudies using Tc99m-labeled agents (e.g., Tc-99m sestimibi or“Cardiolite”) represent a particularly noteworthy diagnostic applicationin dire need of an alternative strategy given the expected contractionof the world's Tc-99m supply over the next 3-5 years. Myocardialperfusion and viability imaging with Tc-99m-related agents is anessential and widely performed procedure, yet no financially feasiblesolution has been offered to replace these Tc-99m-dependent agents.

The use of high-dose parenteral ascorbate as adjunctive chemotherapy forseveral malignancies is the subject of on-going clinical cancer trialsinvestigating this vitamin. Ascorbate MRI as taught herein may thus beuseful to inform the study and application of this chemotherapeutic,coupling high resolution MR imaging to therapeutic administration thatmay enable real-time optimization of delivery and tumor responseassessment.

Ascorbate is now understood to have a pharmacokinetic profile thatresembles vancomycin. Biodistribution of oral ascorbate is under tightcontrol, with plasma concentrations rarely exceeding 200 μM even at oraldoses more than 100 times the recommended daily allowance (Levine M,Conry-Cantilena C, Wang Y, Welch R W, Washko P W, Dhariwal K R, Park JB, Lazarev A, Graumlich J F, King J, Cantilena L R (April 1996).“Vitamin C pharmacokinetics in healthy volunteers: evidence for arecommended dietary allowance”. Proc. Natl. Acad. Sci. U.S.A. 93 (8):3704-9). Ascorbate administered intravenously, however, bypasses thesetight control systems, with plasma concentrations of 10 mM or higherachievable. Plasma concentrations higher than 10 mM are safely sustainedin humans for up to 4 hours with remarkably low toxicity (Hoffer L J.,Levine M., Assouline S., Melnychuk D., Padayatty S J., Rosadiuk K.,Rousseau C., Robitaille L., and Miller W H., Jr., Phase I clinical trialof i.v. ascorbic acid in advanced malignancy. Ann Oncol 19: 1969-1974,2008).

More recent studies have shown than pharmacological ascorbateconcentrations achieved through intravenous infusion results in theformation of ascorbate radicals and the production of hydrogen peroxide(H₂O₂) at concentrations cytotoxic to cancer cells but not normal cells(Chen Q., Espey M G., Sun A Y., Pooput C., Kirk K L., Krishna M C.,Khosh D B., Drisko J., and Levine M. Pharmacologic doses of ascorbateact as a prooxidant and decrease growth of aggressive tumor xenograftsin mice. Proc Natl Acad Sci USA 105: 11105-11109, 2008). Severalpreclinical xenograft studies have demonstrated remarkable growthinhibition with high-dose parenteral ascorbate, and even more recentclinical trials likewise show promising results that also confirm anexcellent safety profile. In preclinical mechanistic studies, theproduction of H₂O₂ is a salient feature. Microdialysis measurementsreveal that H₂O₂ concentrations rise to >100 s μM after high-doseparenteral ascorbate, with both ascorbate and H₂O₂ reaching theirhighest concentrations in tumors and brain regions such as striatum(Chen Q., Espey M G., Sun A Y., Pooput C., Kirk K L., Krishna M C.,Khosh D B., Drisko J., and Levine M. Pharmacologic doses of ascorbateact as a prooxidant and decrease growth of aggressive tumor xenograftsin mice. Proc Natl Acad Sci USA 105: 11105-11109, 2008).

Ascorbate in a pharmaceutically acceptable carrier (e.g., sterile wateror saline) as a formulation for parenteral administration useful forcarrying out the present invention are known, for example for thetreatment of scurvy. Examples of suitable formulations include, but arenot limited to, a sterile solution of ascorbic acid in water forinjection, where each milliliter of water contains: ascorbic acid 500mg, disodium edetate 0.25 mg, sodium hydroxide 110 mg, in water forInjection q.s. pH (range 5.5 to 7.0) adjusted with sodium bicarbonateand sodium hydroxide. Formulations suitable for parenteraladministration may include a pharmaceutically acceptable salt ofascorbate (e.g., sodium salt or meglumine salt), such as a sterilesolution thereof, and may also include pH buffers such as bicarbonate(HCO₃ ⁻) and/or phosphate (PO₄).

The term “pharmaceutically acceptable” as used herein means that thecompound or composition is suitable for administration to a subject toachieve the treatments described herein, without unduly deleterious sideeffects in light of the severity of the disease and necessity of thetreatment.

The present invention is explained in greater detail in the followingnon-limiting examples.

IN VITRO EXAMPLES Ascorbate Enhancement of Spin-Spin Relaxation Rate,1/T₂

Previous studies have reported on the NMR/MRI contrast effects onT₂-weighting arising from exchange of bulk water protons with mobileprotons of low molecular weight solutes and macromolecules (e.g., —NH₂,—OH, —SH, —NH). The contrast effect on 1/T₂ from this proton exchange isdescribed as follows:

$\frac{1}{T_{2}} = {\frac{1}{T_{2a}} + {f_{CR}\left( {P_{b},{\delta\omega}_{b},k,T_{2b},\tau} \right)}}$

Bulk water is related to _(a) and exchangeable protons (e.g., from anascorbate OH group) to _(b). f_(CR) is a closed function with fiveparameters, derived from Carver and Richards and refined by Hill et al.(Carver, J. P.; Richards, R. E. J. General 2-Site Solution For ChemicalExchange Produced Dependence Of T2 Upon Carr-Purcell Pulse Separation J.Magn. Reson. 1972, 6, 89-105; Hills, B. P.; Wright, K. M.; Belton, P. S.N.M.R. studies of water proton relaxation in Sephadex bead suspensionsMol. Phys. 1989, 67, 1309-1326). For the hydroxyl protons of ascorbate,Pb would be the fraction of exchangeable protons, k is the exchange ratebetween exchangeable protons and water protons, δω_(b) is the chemicalshift between hydroxyl and bulk water protons, and T2b is the localspin-spin relaxation time of hydroxyl protons. τ is the inter-pulse(90°-180°) spacing in the T₂-weighted acquisition sequence.

An essential but often neglected parameter influencing proton exchangeon T₂ contrast is the role of exchange catalysis (Liepinsh E and Otting,G Proton exchange rates from amino acid side chains—implications forimage contrast. Magn Reson Med. 1996 35(1): 30-42). The rate constant kfor proton exchange between OH or NH groups and water can be describedby

k=k _(a)[H⁺ ]+k _(b)[OH.]+k _(c)[catalyst]^(y)

K_(a), K_(b) and K_(c) denote the exchange rate constants due tocatalysis by H⁺, OH⁻ and other exchange catalysts, respectively. Theexponent y is 1 or 2 depending on the mechanism of a given exchangecatalyst. The rate constants K_(a) and K_(b) can be calculated in turnby:

$k_{a,b} = {k_{D}\frac{1}{1 + 10^{{pKD} - {pKA}}}}$

where K_(D) is the rate constant for diffusion controlled encounter ofthe proton donor and acceptor (˜10¹⁰ mol⁻¹ s⁻¹), and pK_(D) and pK_(A)are the pK values of the proton donor and acceptor. Although pK_(H3O+)and pK_(OH—)=15.7, K_(c) is more challenging to predict because of thenonlinear dependence of proton transfer on catalyst concentration.Nonetheless, efficient exchange catalysis at neutral pH is attained withat least a moderate difference between pK_(D)−pK_(A) and a significantconcentration of catalytically active acidic or basic forms of theexchange catalysts at physiological pH.

Thus H₂O, despite its high concentration, is an inefficient exchangecatalyst at neutral pH because it's a relatively poor proton donor, withthe pK_(D) and pK_(A) (for H₃O⁺ and OH⁻) are 15.7. On the other hand,recognized exchange catalysts in physiological conditions includeorganic phosphates, carbonates (e.g., bicarbonate, HCO₃ ⁻), andmolecules with carboxyl and amino groups (Liepinsh E and Otting, GProton exchange rates from amino acid side chains—implications for imagecontrast. Magn Reson Med. 1996 35(1): 30-42). As shown below, anotherpowerful catalyst not previously recognized is ascorbate, whichpossesses one hydroxyl group having a favorable pK_(A)=6.75 at the 4position, as well as a disproportionation reaction at equilibrium with apK_(A)=7.0. Thus ascorbate has the potential to not only‘self-catalyze’, but should be an efficient catalyst of proton exchangefor basic hydroxyl groups on sugars and other macromolecules.

FIG. 2 shows a comparison on T₂ enhancement of pure solutions of severalsugars, sugar alcohols, and ascorbate in deionized water at pH 7. Dataare provided from quantitative T₂ mapping at 7T using a RARE FSEprotocol with at least 6 different echo times, at solute concentrationsof 10 and 20 mM. As shown, T₂ relaxivity is roughly a function of thenumber of exchangeable OH protons available on each molecule, withdisaccharides, as predicted, producing proportionally greater contrasteffect than monosaccharides. Noteworthy is the nonlinear dependence onsolute concentration, with relaxivity enhancement decreasing asconcentration is increased, a phenomenon that is likely related toself-association of sugars in pure solutions. The latter is particularlyrelevant to observations described below, where overall T₂ effects areinstead synergistically enhanced when ascorbate and sugars are combinedtogether at higher total solute concentrations. Formulations combiningascorbate with mono or disaccharides provide a means to deliver higherconcentrations of both species in order to increase T₂ contrast effectsfor MR imaging.

FIG. 3A depicts a more detailed demonstration of T₂ effects of pureascorbate solutions at different concentrations at neutral pH. FIG. 3Breveals the marked enhancement of the T₂ effect when ascorbate is in thepresence of only μM (i.e., physiological) concentrations of hydrogenperoxide (H₂O₂), which drives oxidation to dehydroascorbate as well asascorbate radical dismutation. Although H₂O₂ is also considered anexchange catalyst in its own right, the dramatic effect observed onascorbate-mediated 1/T₂ enhancement when H₂O₂ is present at 100-foldless concentration than ascorbate suggests that proton exchange fromH₂O₂—driven ascorbate oxidation/dismutation, rather than direct exchangefrom OH ascorbyl protons, is an important contributory mechanismresponsible for T₂ changes. Further evidence of the contribution fromdehydroascorbate oxidation/dismutation on proton exchange is depicted inFIG. 3C showing that the 1/T₂ enhancement effect is by far the mostsignificant at neutral pH where the reaction rate ofascorbate-dehydroascorbate dispropotionation is also greatest.

Data in FIG. 3D provides the first suggestion that exchange catalysisbetween ascorbate and an acceptor/donor molecule with an appropriate pKcan markedly drive 1/T₂ enhancement change. Data here compare solutionsof ascorbate as sodium salt and as meglumine (aminosugar) salt. Here theT₂ contrast effect (T₂ relaxation in ms) is approximately 4 timesgreater with meglumine ascorbate as compared to either meglumine orascorbate alone in water at neutral pH.

It was subsequently investigated whether the impressive synergisticeffect of meglumine with ascorbate was dependent on chemical associationwith ascorbate as a salting cation (even though in theory the twomoieties should be fully dissociated in water). FIG. 4 reveals thatproton exchange is actually synergized when the ‘salting function’ isperformed by Na⁺ cations, presumably leaving the amine group in additionto the basic OH groups of meglumine to participate in exchange catalysiswith ascorbate. Note that here control T₂ relaxation (ms) values (T₂=840ms) are not shown to better illustrate differences between experimentalgroups.

FIG. 5 summarizes T₂ relaxation data from a series of experimentslooking at the influence of various physiological exchange catalysts onthe contrast effect from ascorbate. Using known serum and extracellularconcentrations of PO₄=2 mM, glucose=5 mM, and HCO₃ ⁻ of 25 mM, withascorbate at 10 mM, T₂ relaxation of each of these moieties was examinedindividually and in combination. As shown, the T₂ relaxation effect ofascorbate alone or with PO₄ in water in modest but in the presence ofphysiological concentrations of either glucose or HCO₃ ⁻ is dramaticallyincreased, with 10 mM ascorbate, (a concentration easily and safelyachieved with parenteral administration) producing a remarkable 50%change in T₂ relaxation. The greatest enhancement is seen with ascorbatein the presence of glucose, HCO₃ ⁻, and PO₄ together at knownconcentrations in vivo. Thus, by simply administering ascorbate i.v.,the T₂ enhancement effect of ascorbate in vivo will be much greater thanwhat might be expected after only looking at ascorbate alone in phantomstudies without physiological exchange catalysts present.

Also predicted from the experiments above is the possibility thatformulation of ascorbate with other sugars that are not normally presentin vivo may further catalyze the ascorbate contrast effect, FIG. 6, forexample, demonstrates additional synergism when meglumine is added to asolution of sodium ascorbate at equivalent concentration (20 mM) andinto a background of 2 mM PO₄, 25 mM HCO₃ ⁻. Data show comparison withor without physiological concentrations (5 mM) of glucose, as well asthe effect of meglumine alone added to the physiological catalysts. Asseen the greatest contrast effect is observed when all moieties arecombined. One implication therefore is that higher contrast effects maybe achievable by combining different exchange catalysts with each otherthus limiting the concentration of any one exogenously administeredspecies.

FIG. 7 summarizes data extending this concept, testing potentialsynergisms when Na ascorbate and meglumine are formulated with othermono and disaccharides and sugar alcohols. As shown the contrast effectsare dramatic with each potential formulation. In FIG. 8, the controlsolution (2 mM PO₄ and 25 mM HCO₃ ⁻) to better illustrate thedifferences in contrast changes between groups. The strongest effectthus observed is when ascorbate and meglumine are combined with thecommon disaccharide sucrose, thus suggesting a promising candidateformulation (i.e., ascorbate/meglumine/sucrose) for MRI using onlymoieties that may all be safely administered parenterally.

In Vivo Example 1 Normal Brain Perfusion and Metabolic Change

In vivo ascorbate T2 contrast changes were determined following highdose parenteral ascorbate (2 g/kg, right IJ iv injection). FIG. 9A showsa conventional single slice axial FSE T2WI image through the midbrain ofa normal C57 black mouse, and the two images on the right demonstrate a‘first pass’ extraction of contrast change during and followingascorbate administration i.v. T2 signal in brain tissue immediatelyfollowing, and 10 minutes after, ascorbate administration is acquiredand then subtracted from the T2 brain signal acquisition pre ascorbateadministration. Since ascorbate produces a decrease in signal intensity,subtraction from the higher signal pre-dose scan results in a netpositive ‘map’ of flow-through perfusion (blood flow) through braintissue. At 10 minutes, the perfusion effect has nearly resolved andearly signal intensity changes related to tissue uptake are beginning tobe observed. FIG. 9B shows the signal changes due to tissue uptake ofhigh-dose ascorbate. Color-lute maps of signal intensity are notsubtracted from the pre-dose scan and therefore show the expecteddecreases in T2 signal over time, maximized between 30-60 min in normalC57 mice.

In Vivo Example 2 Focal Cerebral Hypermetabolism in Association withNeocortical Spreading Depression

FIG. 10 shows ascorbate T2 enhancement in a rodent model of neocorticalspreading depression. Spreading depression (SD) is an experimentallyreproducible pathophysiological phenomenon of CNS tissues originallydescribed 60 years ago by Loao. After a focal region of cortex reaches acritical threshold of ionic perturbation, a massive spreading wave ofcellular depolarization may begin and spread through gray matter tissue,but remain confined to the gray matter zone in which it was induced, notcrossing white matter pathways. If the induction mechanism (e.g., alocal high concentration of applied potassium chloride) is continuous tothe same region, these waves of spreading depression will recur onceevery 8-10 minutes and last over a 2-3 hour period. Marked changes inbrain metabolism accompany SD, and since no histologically detectableneuronal injury is present after SD, these metabolic changes parallelmetabolic fluxes in non-ischemic, hyperexcitable brain tissue such asepileptogenic foci.

In the above experiment, the lower row of images shows a tinycraniotectomy with gelfoam (red arrow) soaked in a high concentration ofKCl, which diffuses locally into the adjacent parietal cortex. Thecraniectomy site is 1 mm posterior to bregma, a skull landmarkrepresenting the posterior third of the underlying brain. The above tworows show T2 images and quantitative color lute T2 maps of signal changein rodent brain that are 3 and 4 mm anterior to bregma, that is, distantfrom the SD induction site. T2 signal changes in the anterior slicesdemonstrate clear T2 asymmetry in the right cerebral cortex as comparedto the left (again, SD remains confined to the right hemisphere). Thesemarked cortical signal changes are consistent with the knownhypermetabolic activity that occurs with SD, as also observed with¹⁸F-FDG PET, and with direct microdialysis and metabolomicdeterminations. Of note, the opposite observation (focally increased T2signal) is seen directly under the craniectomy site itself (row three),consistent with localized edema (increased free water) at the site ofKCl infusion.

In Vivo Example 3 Cardiac Perfusion and Metabolic Imaging

Perfusion and viability cardiac imaging with parenteral ascorbate wasassessed. FIG. 11A depicts the two primary imaging planes, coronal andaxial, for rat heart imaging at 7T. Retrospective gating withrespiratory coupling was employed to collect images at 7T. Theacquisition sequence is moderately T2-weighted and can be furtheroptimized enhance the contrast effect. FIG. 11B shows transient decreasein T2 signal intensity throughout the left ventricle with initial bolusof ascorbate injection i.v. After the initial bolus for first pass flowor ‘perfusion imaging’ quantitative T2 maps using variable flip anglesshow gradual T2 contrast change in heart tissue reflecting ascorbateuptake. Only viable, metabolically active cells will take up ascorbate.

In Vivo Example 4 T2 Contrast Changes in Guinea Pigs FollowingIntravenous Administration of Three Different Formulations of Ascorbate

We examined T2 contrast changes in whole brains of lightly anesthetizedguinea pigs at 7T. Since guinea pigs share humans' inability tosynthesize ascorbate endogenously, MRI effects in this model may be morepredictive of MRI changes in patients. Ascorbate was administeredparenterally via femoral or jugular vein access using controlledinfusion for a total dose of 2 g/kg over 60 minutes. MRI was performedfor 90 minutes.

FIG. 12A shows Fast spin echo (FSE) T2 images before and after 60 minslow infusion of ascorbate show dramatic signal intensity differencesthroughout the brain parenchyma.

In FIG. 12B and FIG. 12C, normalized signal intensity changes andquantitative relaxivity measurements are shown for both guinea cerebralcortex (Cx) and basal ganglia (BG) after administration of threedifferent ascorbate formulations: (1) 100% sodium ascorbate; (2) 50%sodium ascorbate and 50% meglumine ascorbate; and 3) 100% meglumineascorbate. In FIG. 12B, signal intensity changes are greatest at eachtime point during and following administration of the second formulation(2) consisting of 50% Na AA: 50% Meg AA, with observed cortical FSE T2intensity decreases exceeding 40%. Calculated T2 relaxivity values inFIG. 12C also show a greater than 10% from baseline with formulation(2), with maximal values statistically greater than either formulation(1) or (3). On conventional FSE T2 weighted images, signal intensitychanges with Meg AA (3) are also noted to be greater than those observedwith sodium ascorbate (1) at nearly every time point, however T2relaxivity calculations do not show statistical differences betweenthese latter two formulations.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of enhancing a magnetic resonance imaging (MRI) image of a body or body region such as an organ or organ region in a subject, comprising: parenterally administering a composition comprising ascorbate or a pharmaceutically acceptable salt thereof to said subject in an MRI image-enhancing amount, wherein said composition comprises a spin-spin exchange catalyst; and then generating, by MRI of the subject, an image of said body or body region, whereby said composition enhances the MRI image.
 2. The method of claim 1, wherein said body region is a head, neck, thorax, abdomen, pelvis, limb(s), muscle, fat, or bone.
 3. The method of claim 1, wherein said organ comprises an adrenal gland, pituitary, thymus, corpus luteum, retina, brain, spleen, lung, testicle, lymph nodes, liver, thyroid, small intestinal mucosa, leukocytes, pancreas, kidney, or salivary gland tissue.
 4. The method of claim 1, wherein said body region is a brain.
 5. The method of claim 1, wherein said body region is a heart.
 6. The method of claim 1, wherein said ascorbate or pharmaceutically salt thereof is administered in an amount of from 0.02 grams per kilogram subject body weight, up 40 grams per kilogram subject body weight.
 7. The method of claim 1, wherein said administering step is carried out by intravenous administration.
 8. The method of claim 1, wherein said administering step is carried out by intraperitoneal administration.
 9. The method of claim 1, wherein said image comprises a T₂ weighted image.
 10. The method of claim 1, wherein said image comprises a metabolism image.
 11. The method of claim 10, wherein said metabolism image is a tumor metabolism image or a brain metabolism image.
 12. The method of claim 1, wherein said image comprises a perfusion image.
 13. The method of claim 12, wherein said perfusion image is a cardiovascular perfusion image.
 14. The method of claim 1, wherein the generating is performed during, or up to 120 minutes after, parenterally administering said composition.
 15. The method of claim 1, wherein said composition comprises sodium ascorbate, meglumine ascorbate, or a mixture thereof.
 16. The method of claim 1, wherein said spin-spin exchange catalyst is a sugar or a sugar alcohol.
 17. The method of claim 1, wherein said composition comprises a mixture of meglumine ascorbate and sodium ascorbate provided in a molar or millimolar (mM) ratio of from 10:90, 20:80, 30:70, or 40:60, up to 90:10, 80:20, 70:30, or 60:40 (meglumine ascorbate:sodium ascorbate).
 18. A method of enhancing a magnetic resonance imaging (MRI) image of a body or body region such as an organ or organ region in a subject, comprising: parenterally administering a composition comprising ascorbate or a pharmaceutically acceptable salt thereof to said subject in an MRI image-enhancing amount, wherein said composition comprises meglumine; and then generating, by MRI of the subject, an image of said body or body region, whereby said composition enhances the MRI image.
 19. The method of claim 18, wherein said body region is a head, neck, thorax, abdomen, pelvis, limb(s), muscle, fat, or bone.
 20. The method of claim 18, wherein said organ comprises an adrenal gland, pituitary, thymus, corpus luteum, retina, brain, spleen, lung, testicle, lymph nodes, liver, thyroid, small intestinal mucosa, leukocytes, pancreas, kidney, or salivary gland tissue.
 21. The method of claim 18, wherein said body region is a brain.
 22. The method of claim 18, wherein said body region is a heart.
 23. The method of claim 18, wherein said ascorbate or pharmaceutically salt thereof is administered in an amount of from 0.02 grams per kilogram subject body weight, up to 40 grams per kilogram subject body weight.
 24. The method of claim 18, wherein said administering step is carried out by intravenous administration.
 25. The method of claim 18, wherein said administering step is carried out by intraperitoneal administration.
 26. The method of claim 18, wherein said image comprises a T₂ weighted image.
 27. The method of claim 18, wherein said image comprises a metabolism image.
 28. The method of claim 18, wherein said image comprises a perfusion image.
 29. The method of claim 18, wherein the generating is performed during, or up to 120 minutes after, parenterally administering ascorbate or pharmaceutically acceptable salt thereof.
 30. The method of claim 18, wherein said composition comprises a mixture of sodium ascorbate and meglumine ascorbate in a molar ratio of from 20:80 to 80:20 of meglumine ascorbate:sodium ascorbate. 